Hydrophilic polypeptides and their preparation



Unitw tes. Patent v g 3,004,004 l HYDROPHIIJC POLYPEPTIDES' AND THEEPREPARATION Charles J. Fox, Rochester, N.Y., assignor to Eastman KodakCompany, Rochester, NtY.,- a corporation. of New Jersey No Drawing.Filed Marr-26; 1958, Ser. No. 723,930 2 Claims. (Cl. 260-78)- Thisinvention relates to hydrophilic polypeptides prepared by treatingpolypeptides of benzyl glutamic carboxy anhydride while in solution indichloroacetic acid with hydrogen bromide and the products thusobtained.

Hydrophilic polypeptides are potentially valuable liecause of theirsimilarity in structure to naturally occurring proteins. For instance,these products can be useful for replacing gelatin such as inphotographic products or as peptizers in the preparation of silverhalide.

The preparation of synthetic' polypeptides of high molecular weight,such as of a degree of polymerization greater than 1,000 by variousmethods has been suggestedsuch as by the polymerization of amino acidsor their suitable derivatives serving asmonomers. Attempts to polymerizenatural amino acids were recorded even before the existence of thepeptide compounds had been clearly established. The main object of theseearly attempts was synthesis from the structural units of the proteinmolecule; For instance, a waterin'soluble polymer having propertiesresern ring those of a horny protein was obtained by mid ing glycine inglycerol. The product obtained, however, had no definite constitutionand apparently represented mixtures of materials.

In many of the well-known methods of peptide" syn. thesis, polypeptidesof relatively low molecular weight were obtained. The monomers whichhave been used for the synthesis of poly amino acids have included thefollowing groups: free amino acids, esters of amino acids, esters andazides of peptides, N-carboxy-y-amino acid anhydrides. Variousconditions have been suggestedf'o'r carrying out the reaction such ashaving the monomers in solution in room temperature or in solvents athigh temperature to obtain polypeptides of. high degrees ofpolymerization. It has often been desirable to carry out thepolymerization of these derivatives in the presence of a basic catalyst.It has been considered desirable to employ amino acid. derivatives of atype in. which. a protective group such as benzyl was present to inhibitsecondary reactions. Some of the initiators or catalysts which have beensuggested particularly forthe preparation of high molecularpoly-"y-benzyl-L glutamates by polymerizing 'y-benzyl glutamic-N-carboxyanhydride have been triethyl amine, diethyl amine, sodium methoiride,hexyl'amine, sodium hydroxide, or sodium borohydride. Thepolymerizations have been carried out by dissolving'y-benzyl-N-carboxy-L-glutamic anhydride in a suitable solvent such asdioxane and then carrying out the polymerization under the influence ofthe initiator or catalyst.

'y benzyl glutamic-N-carboxy anhydride has been-copolymerized with otheramino acid N-carboXy anhydrides and the copolymers prepared remainsoluble in dioxane when the molar ratio of the first monomer to thesecond benzyl group. Up to now 'a convenient and practical method ofremoving this blocking benzyl group has not been available. Methods havebeen described in which solubility of the polymer in glacial acetic acidwas required. However, polypeptdies' of high degrees of polymerizationobtained from polymerizing 'y-benZyleL-gluacid. and many other organicsolvents.

which degradation may seriously detract from the fibrous character ofthe polymer and increase brittleness even though free carboxyl and aminegroups. are formed therein.

One object of my inventionisto provide a method for supplying freecarboxyl groups to polypeptides-prepared by the polymerization. of.-benzyl-glutamic-N-carboxy: anhydrides. Another object. of. myinvention is topro vide a method} for supplying free carboxyl groups topolypeptides in which. degradation of the polypeptide chain is avoided.vA further object or my invention is tov provide a methodofv preparing.polypeptides of. high molecular. weights, the sodium and. ammonium saltsof which are soluble in: water. Other objects of my invention willappear herein. a

I havefound that polypeptides obtained bythe polymerization of aminoacid monomers in which at least 50% of the monomer mixture is'y-benzyl-glutamic-N- carboxy anhydride are readily converted to a formwherein free carboxyl groups are present therein if the polymericmaterial is dissolved in dichloroacetic acid and the resulting solutionis treated with anhydrous hydrogen bromide. The resulting product isreadily converted to the sodium or ammonium salts thereof by treatmentwith a sodium or ammonium base and the resulting salts obtained arereadily soluble in water. I have found that by this procedure the degreeof polymerization of the polypeptide is not appreciably reduced and aproduct of high molecular weight and good viscosity is obtained. Toobtain products having. useful properties, it is desirable that thepolypeptide have an intrinsic viscosity in dioxane I of at least 0.5.Upon treatment of the polypeptide, in accordance with my invention, aproduct. is obtained, the degree of, polymerization of which is notappreciably diminished and the sodium or ammonium salt of which is watersoluble and useful as addenda to or as a substitute for gelatin inaqueous solutions thereof, particularly for photographic purposes.

In preparingv polypeptidesfrom 'y-benzyl-glutamicrN- carboxyanhydride,itis. desirable that that material coiistitute at least 50% of theprotein derivative employed in the polymerizationprocedure. Thepolymerization may. be carriedout' using the'y-benzyl-glutamic-N-carboxy anhydride as the entire amino acidderivative therein or that material may be present in any molar ratiostarting with at least equal parts with other amino acid carboxyanhydrides such as those of leucine or glycine. It is preerred that thelaevo form of the amino acid derivatives be employed, although a mixtureof the laevo and dextro forms of the amino acid derivative can be used.

After the polymer is prepared, it is treated in accordance with myinvention which involves dissolving in thchloroacetic acid and applyinganhydrous hydrogen bromide thereto over a short period of time tointroduce carboxyl groups therein, pteietab-ly'with temperature com trolbecausethe replacement of benzyl group with carboxyl is an exothermicreaction. The hydrogen bromide is conveniently supplied to the reactingmaterialby continuously introducing the gas into the solution over theperiod of the reaction at a rate which is'found to'be most convenient bythe operator of the procedure. z'Hydrogen bromide should always bepresent in the dichloro- V acetic acid solution over the course of thereaction.

The following examplesillustrate both the polymerization ofy-benzyl-glutamic-N-carboxy anhydride and the Patented: Oct. 10,. 1961:.

replacement of benzyl therefrom in accordance with my invention.

Example 1.-Plymerization of 'y-benzyl-L-glutamic-N- carboxy anhydride 20grams of 'y-benzyl-L-glutamic-N-carboxy anhydride was dissolved in 250ml. of dioxane and 0.05 grams of triethylarnine was added thereto. Themass was allowed to stand overnight. It was then poured into water and aprecipitate of fibrous polymer was obtained.

Example 2 A solution of 10 grams of poly-'y-benzyl-L-glutamic acid asprepared by the preceding example and having an intrinsic viscosity indioxane of 0.58 was dissolved in 50 ml. of dichloroacetic acid. Thissolution was cooled in an open vessel, placed in an ice bath, andanhydrous hydrogen bromide was bubbled therethrough for 20 rnin utes atthe rate of 1 liter of gas every 10, minutes. The product was separatedfrom the mass by precipitation in water and was washed thoroughly withwater and with ether to remove hydrogen bromide, dichloroacetic acid,and benzyl bromide. A white fibrous polymer which was soluble indilutealkali was obtained in 96% .yield. The polymer product obtained gave anintrinsic viscosity in dioxane of 0.74 and a carboxyl analysis of 29.2%.

Example 3 14 grams of poly-'y-benzyl-L-glutamic acid having an intrinsicviscosity in dioxane of 0.42 was dissolved in 200 ml. of dioxane. Therewas supplied to the solution, anhydrous hydrogen bromide at the rate of1 liter of gas every 10 minutes, for 1 /2 hours at room temperature andfor 1 hour at 40-50 C. After this treatment, the polymer remainedinsoluble in dilute alkali. The solution saturated with hydrogen bromidewas allowed to stand overnight at room temperature. The polymer obtainedwas separated from the mass by precipitation in water and was thoroughlywashed as described in the preceding example. A white fibrous polymerwas obtained which was soluble in dilute alkali. This product gave anintrinsic viscosity in dioxane of 0.46 and a carboxyl analysis of 22.4%.This example as compared with the preceding exhibits the advantages ofusing dichloraceti'c acid as the solvent in the hydrogen bromidetreatment as compared to dioxane. When dichloracetic acid is used thereaction rate is improved over that of dioxane.

'Example 4.--Copolymerization of 'y-benzyl-L-glutamic- N-carboxyanhydride and L-leucine-N-carboxy anhydride in a 1 :1 molar ratioExample 5 The polymer obtained in the preceding example was dissolved in50 ml. of dichloroacetic acid which solution was cooled in an ice bathfor 30 minutes during which anhydrous hydrogen bromide was suppliedthereto at the rate of 1 liter of gas every minutes. The resultingproduct was separated therefrom by precipitation into water whichprecipitate was thoroughly washed with water and with ether and wasdried. A white fibrous polymer, soluble in dilute alkali and having anintrinsic viscosity in dioxane of 2.22, was obtained quantitatively.

Example 6.Cop0lymerization of 7-benzyl-L-glutamic- N-carboxy anhydrideand L-leucine-N-carboxy anhydride in a 2:1 molar ratio hours at roomtemperature, an additional 0.01 gram of triethylarnine was added and themass stood for an additional 24 hours. The product thus obtained wasseparated from the mass by precipitation into water. 4.5 grams of awhite fibrous polymer having an intrinsic viscosity of 1.43 in dioxanewas obtained in 82% yield.

Example 7 The polymer obtained in the preceding example was dissolved in50 ml. of dichloroacetic acid. The solution was cooled on an ice bathand was treated with anhydrous hydrogen bromide for 30 minutes at therate of 1 liter of gas every 10 minutes. The product resulting wasisolated by precipitation into water and was then washed thoroughly withwater and ether and dried. A white fibrous polymer having an intrinsicviscosity of 1.85 in dioxane was obtained in 94% yield.

Example 8.-C0polymerlzati n of q-benzyl-L-glutamic- N-carboxy anhydrideand L-leucine-N-carboxy anhydride in a 3:1 molar ratio 7.89 grams of'y-benzyl-L-glutamic-N-carboxy anhydride and 1.57 grams ofL-leucine-N-carboxy anhydride was dissolved in ml. of dioxane. There wasthen added 0.01 gram of triethylarnine and after standing 24 hours anadditional 0.01 gram of triethylarnine was added. The mass then stoodfor 24 hours longer and the product was then separated by precipitationinto water. 6.3 grams of a white fibrous polymer having an intrinsicviscosity of 1.18 in dioxane was obtained in 82% yield.

Example 9 The polymer obtained in the preceding example was dissolved in50 ml. of dichloroacetic acid. The solution was cooled on an ice bathand treated with anhydrous hydrogen bromide for 30 minutes by bubblingthe gas through the mass at the rate of 1 liter thereof every 10minutes. The product obtained was separated from the mass byprecipitation into water following which the product was washedthoroughly with water and ether and dried. A white fibrous polymersoluble in dilute alkali and having an intrinsic viscosity of 1.59 indioxane was obtained quantitatively. This product showed a carboxylcontent upon analysis of 27.2%.

Polypeptides may be obtained by copolymerizations in which the benzylglutamic carboxy anhydride is present in a minor proportion. However,those products are usually insoluble in dioxane and in dilute alkali.The preparation of products of this type are illustrated by thefollowing examples:

Example 10.Copolymerization of 'y-benzyl-L-glutamic- N-carboxy anhydrideand L-leucine-N-carboxy anyhydride in a 1:2 molar ratio 5.26 grams of'y-benzyl-L-glutarnic-N-carboxy anhydride and 6.28 grams ofL-leucine-N-carboxy anhydride were dissolved in 100 ml. of dioxane and0.01 grams of triethylarnine was added. The mass stood for 24 hours atroom temperature whereupon an additional 0.01 grams of triethylarninewas added. The mass was allowed to stand for an additional 96 hours andan almost solid gel was obtained. The gel was diluted to 250 ml. withdioxane and the polymer therein was isolated by precipitation intowater. 8.0 grams of white fibrous polymer was obtained representing a91% yield. This product swelled in, but was not soluble in dioxane.

Example 11 The product obtained in accordance with the preceding examplewas dissolved in 50 ml. of dichloroacetic acid and the solution wascooled on an ice bath and was treated with anhydrous hydrogen bromidefor 2 hours at the rate of 1 liter of gas every 10 minutes. The polymerobtained was precipitated into water, washed thoroughly in water and inether and was dried. A white fibrous polymer was obtained in 95% yield.This polymer was insoluble in dilute alkali but dissolved when 10% ofethanol was added. The polymer was insoluble in dioXane but swelledtherein.

I claim:

1. A process for preparing a hydrophilic polypeptide which comprisesdissolving a high molecular weight polyy-benzyl-L-glutanfic acid, havingan intrinsic viscosity in dioxan of at least 0.42, in dichloracetic acidand treating the solution thus obtained with anhydrous hydrogen bro mideunder cool conditions until the debenzylated polypeptide is soluble indilute alkali.

References Cited in the file of this patent UNITED STATES PATENTSMacDonald Mar. 3, 1953 OTHER REFERENCES Sela et al.: J.A.C.S., vol. 78,pp. 746-751 (1956).

Idelson et al.: I.A.C.S., vol. 80, p. 4631 (1958).

'Bamford et al.: Synthetic Polypeptides (1956), publ. by Academic PressInc., NY.

Noller: Textbook of Organic Chem, 1951, page 146, publ. by W. B.Saunders Co., Philadelphia, Pa.

1. A PROCESS FOR PREPARING A HYDROPHILIC POLYPEPTIDE WHICH COMPRISESDISSOLVING A HIGH MOLECULAR WEIGHT POLYY-BENZYL-L-GLUTAMIC ACID, HAVINGAN INTRINSIC VISCOSITY IN DIOXAN OF AT LEAST 0.42, IN DISCHLORACETICACID AND TREATING THE SOLUTION THUS OBTAINED WITH ANHYDROUS HYDROGENBROMIDE UNDER COOL CONDITIONS UNTIL THE DEBENZYLATED POLYPEPTIDE ISSOLUBLE IN DILUTE ALKALI.